Project Leader: JT Niones
Recognizing the impact, importance and potential utilization of microbial, invertebrates and plant resources from the rice environment, PhilRice has been studying, evaluating and promoting the use of beneficial microbes ranging from fungi, bacteria and actinomycetes either as potential biological control agent of specific rice pest or as plant growth promoter.
Pure isolates of rice pathogens are regularly cultured and utilized in the evaluation of rice plant breeding materials for disease resistance. Moreover, PhilRice has recently renewed its interest in Azolla technology and other N-fixing systems in support to the organic agriculture program.
Along with the increasing collection of these beneficial microbial and non- microbial resources at PhilRice, is the pressing concern to provide a reliable and safe preservation and storage protocol for these genetic resources. Physiological or genetic damage to economically important strains could potentially result in considerable loss of investment in a research and product development program.
The project aims to: (1) develop conservation and preservation strategies for beneficial microbes, invertebrates and plant resources from the rice environment to ensure their physiological and genomic integrity and quality, for research and development, and public utilization purposes; (2) to establish management system that facilitates record-keeping, utilization, distribution and exchange of these genetic resources.
Conservation and management of azolla species CLC Mondejar and GO San Valentin
When the National Azolla Action Program (NAAP) was implemented in 1982, selected azolla varieties were distributed in different regions of the country. These varieties of azolla were identified to be suitable in the area of dispersal. Nowadays, azolla thrives in rice community in small population only and some plants can be found in irrigation canals instead on rice paddies. Only few farmers are aware of the importance of azolla in rice farming and sustain the utilization of azolla.
The PhilRice has the capability to continue R&D activities on azolla and other N-fixing systems in support to the organic agriculture program of the Department of Agriculture (DA). As a leader of rice R&D in the country, PhilRice initiated the recovery of the original accessions and continue the selection and hybridization to produce superior strains. The researches on azolla in PhilRice have been focusing mainly on how these plant would
become a stable part of rice farming system. PhilRice makes the azolla technology available by propagating fresh biomass and other inoculum for distribution to farmers.
The re-establishment of azolla technology by PhilRice requires the development of living azolla reference collections. The reference collection will serve as a source of inoculum for (1) continuous production of azolla and (2) basic and applied researches on azolla. The main goal of the establishment of azolla germplasm collections in PhilRice is to conserve the Philippine azolla indigenous species and accessions widely adapted in the Philippine condition for utilization.
Highlights:
• Two methods are used in conserving the azolla in PhilRice Los Baños. These are the in vitro method and soil-and-water medium. Both of these methods use the vegetative means of propagation. The in vitro method uses the protocol of Watanabe et al. 1992 and nutrient solution as the medium (Figure 13). In soil-and-water medium, the azolla are grown in plastic trays filled with paddy soil with at least 2cm high water level (Figure 14).
• A system of accessing the different azolla strains in PhilRice azolla germplasm collections was made. Figure 15 shows the entry of new accessions in the germplasm while figure 16 shows the process of acquiring azolla from PhilRice.
• A method to characterize azolla based on growth rate was standardized. Azolla mexicana # 2024 or the UPLB Hybrid 1 was used as the test strain. Figure 17 shows the differences in the growth response of four azolla strains under Los Baños, Laguna condition.
Table 18. List of azolla accession available at PhilRice Azolla Germplasm Collection as of December 31, 2015 for distribution.
No Accession
no. Other Code Donor AcquisitionDate of Remarks 1 prri-az-001-001 PI 0072 IRRI 7/8/2014,
8/19/2014 NAAP recommended strain 2 prri-az-001-002 CA 3002 IRRI 7/8/2014,
8/19/2014
NAAP recommended strain
3 prri-az-001-003 CA 3005 IRRI 7/8/2014, 8/19/2014
NAAP recommended strain
4 prri-az-001-004 MI 4018 IRRI 7/8/2014, 8/19/2014
NAAP recommended strain
5 prri-az-001-005 ME 2024 IRRI 7/8/2014,
8/19/2014 NAAP recommended strain 6 prri-az-001-006 ME 2028 IRRI 7/8/2014,
8/19/2014
NAAP recommended strain
7 prri-az-001-007 PP 7001 IRRI 7/8/2014, 8/19/2014
NAAP recommended strain
8 prri-az-001-008 PP 7004 IRRI 7/8/2014, 8/19/2014
NAAP recommended strain
9 prri-az-001-010 PI 0005 IRRI 7/8/2014,
8/19/2014 NAAP recommended strain 10 prri-az-001-011 ME 2002 IRRI 7/8/2014,
8/19/2014
NAAP recommended strain
11 prri-az-001-023 PRRI 14-001 CLC Mondejar 8/4/2014 Brgy. Taytay, Majayjay, Laguna GO San
Valentin
12 prri-az-001-024 PRRI 14-007 CLC Mondejar 8/4/2014 Brgy. Tinamnan, Lucban, Quezon GO San
Valentin
13 prri-az-001-029 PRRI 14-013 CLC Mondejar 10/3/2014 Brgy. Basud, Polangui, Albay GO San
Valentin
14 prri-az-001-030 PRRI 14-004 CLC Mondejar 10/3/2014 Brgy. 4, Tugawe-Site, Malilipot, Albay
GO San Valentin
15 prri-az-001-031 PRRI 14-005 CLC Mondejar 10/3/2014 Brgy. Balabag, Milaor, Camarines Sur
GO San Valentin
16 prri-az-001-032 PRRI 14-006 CLC Mondejar 10/3/2014 PhilRice Bicol Station, Batang, Ligao City
GO San Valentin
17 prri-az-001-065 PRRI 15-009 TC Fernando 2015 Adams, Camarines Norte (mexicana)
18 prri-az-001-066 PRRI 15-010 TC Fernando 2015 Adams, Camarines Norte (pinnata) 19 prri-az-001-067 PRRI 15-007 CLC Mondejar 2015 Brgy. 4, Paete, Laguna
20 prri-az-001-068 PRRI 15-008 CLC Mondejar 2015 Talavera St., Pakil, Laguna
Figure 13. In vitro conservation of azolla.
Figure 14. Conservation in soil-and-water medium at PhilRice Azolla Nursery in PhilRice-Los Baños.
Figure 15. The flow in the entry of new accession to the azolla collection.
Figure 16. The flow in acquiring accessions from the azolla collection of PhilRice.
Figure 17. Growth rate of four azolla strains (ME 2024, ME 2027, ME 2030 and ME 2033) at linear phase of growth using the standard protocol in
characterizing azolla.
Conservation and management of biocontrol agents GF Estoy Jr. and BM Tabudlong
The use of biocontrol agents provide an alternative pest control measure to reduce pest population below damaging level. It has shown promise in managing the rice pests’ problem (PhilRice, 1995). By their specific nature, it is likely to cause less harm if any to beneficial organisms than chemical pesticides. During outbreak of pests, there is a great demand of biological control agents (BCA’s) production of adequate quantities of high quality inoculums.
Conservation biological control is a strategy that seeks to integrate beneficial insects back into crop systems for natural pest control. This strategy is based upon ongoing research that now demonstrates a link between the conservation of natural habitat and reduced pest problems on farms.
Conservation strategies for BCA’s are necessary in order to use them and optimize its efficacy, stability, safety and ease of application.
The study aims to develop management strategies to conserve, preserve BCA’s and evaluate the efficacy of these conservation strategies.
Highlights:
• Ten isolates of Beauveria bassiana (Table 19), 10 isolates of Metarhizium anisopliae (Table 20) and one isolate of Paecilomyces sp. are being maintained in potato dextrose agar.
Pure cultures of these entomopathogens were overlaid with mineral oil prior to their storage inside a refrigerator.
• The viability of Beauveria bassiana, Metarhizium anisopliae and Paecilomyces sp. preserved using mineral oil was evaluated after four and six months in storage inside a
refrigerator. Upon culture revival, mycelia of the fungal isolates grew well in culture medium and thus remained viable (Table 21).
• The virulence of the preserved cultures of entomopathogens was evaluated against major rice pests. Strains of B. bassiana, 6 months in storage in mineral oil, were pathogenic to rice bug adult with a mortality ranging from 43% to 100% at 10 days post treatment (Table 22). On the other hand, strains of M. anisopliae caused 60 to 83% mortality on a population of white stemborer larvae, 56 to 93% against rice bug and 70 to 86% mortality on rice black bug (Table 23).
Table 19. List of different strains of Beauveria bassiana in potato dextrose agar slants.
No. Code Name
Insect Host Place Collected 1 Bb.#01 Sweet potato
weevil
ViSCA, Baybay, Leyte 2 Bb.#02 Coleoptera ViSCA, Baybay, Leyte
3 Bb.#21 Rice bug Mahanub, Gigakit, Surigao Del Norte
4 Bb.#22 Rice Bug Prosperidad, Agusan Del Sur 5 Bb.#27 Rice bug Trento, Agusan Del Sur 6 Bb.#33 Undetermined
larvae RTRomualdez, Agusan Del Norte 7 Bb.#41 Coccinellid beetle Alipao, Alegria, Surigao Del Norte 8 Bb.#42 Coccinellid beetle Gigakit, Surigao Del Norte 9 Bb.#49 Rice bug RTRomualdez, Agusan Del Norte 10 Bb.#52 Rice bug RTRomualdez, Agusan Del Norte
Table 20. List of different strains of Metarhizium anisopliae in potato dextrose agar slants.
No. Code Name
Insect Host Place Collected 1 Ma.#01 Sweet potato
weevil
Tiaong, Quezon
2 Ma.#3 Rice black bug Capatungan, Trento, ADS 3 Ma.#5 Rice black bug Bual , North Cotabato 4 Ma.#6 Rice black bug Basilisa, RTR, ADN 5 Ma.#15 Rice black bug Ponyente, Gigakit, SDN 6 Ma.#16 Rice black bug Esperanza, Prosperidad,
ADS
7 Ma.#17 Rice black bug Kitcharao, ADN
8 Ma.#19 Rice black bug San Andres, Bunawan, ADS
9 Ma.#20 Rice black bug San Francisco, ADS 10 Ma.#116 Rice black bug Basilisa, RTR, ADN
Table 21. Preservation of B. bassiana, M. anisopliae and Paecilomyces sp.
isolates in mineral oil.
Fungal biocontrol Code Effectivity of Fungal Growth
Agents Name After 4
months
After 6 months Beauveriabassiana Bb.#42 Viable Viable
Bb.#001 Viable Viable
Bb.#33 Viable Viable
Bb.#208 Viable Viable
Metarhizium anisopliae Ma.#19 Viable Viable
Paecilomyces sp. Pf.#9 Viable Viable
Table 22. Mortality (%) of the rice bug at 7 days post treatment with different isolates of B. bassiana under laboratory condition.
Fungal Isolates
Rice Bug adult
Mortality (%) LT50(days)
Bb.27 93.33 a 4.8
Bb.33 100.00 a 4.9
Bb.49 60.00 bc 5.8
Bb.22 66.67 bc 5.5
Bb.52 50.00 c 7.5
Bb.21 80.00 ab 6.2
Bb.42 50.00 c 7.5
Bb.02 53.33 bc 7.2
Bb.01 43.33 c *
Control 0
Table 23. Mortality (%) of 2-day old white stemborer larvae, rice bug and rice black bug, 7 days post treatment with different isolates of M. anisopliae under laboratory condition.
Fungal Isolates
Mortality (%)
WSB RB RBB
Ma.SanFrancisco 80.00 ns 60.00 b 86.67 ns
Ma.Surigao 83.33 73.33 ab 76.67
Ma.Bunawan 86.67 56.67 b 70.00
Ma.Esperanza 63.33 63.33 b 80.00
Ma.Buenavista 66.67 66.67 ab 83.33
Ma.Capatungan 80.00 70.00 ab 80.00
Ma.Gigakit 73.33 83.33 ab 83.33
Ma.Sogod 60.00 56.67 b 76.67
Ma.Tiaong 80.00 93.33 a 86.67
Control 0 0 0
Figure 18. Preservation of fungal biological control agents in oil form.
Conservation and management of microbial agents JT Niones and FR Sandoval
The role and impact of microorganisms on agronomically important crops depend on their interaction with their host plant. Negative interaction of microorganisms with their host resulted to diseased plants and significant crop loss while positive host- microorganism interaction can improve crop nutrition and the ability of crops to resist biotic and abiotic stress. With the increasing awareness of the undesirable human and environmental effects of the use of inorganic fertilizers, herbicides and pesticides, PhilRice thorugh its R&D programs had long recognized that beneficial microbes provide an alternative strategy to combat limiting soil nutrient and the destructive effects of weeds and pests on crops.
Maintaining and preserving fungal cultures are not only essential on systematics and biodiversity studies but also in ensuring the quality of microbial agents especially for commercialization and public utilization purposes. Preservation methods of potentially important isolates for agrobiological applications have to be optimized early in the development process of a product so as to avoid potential economic and scientific loss in the event of deterioration of a production strain.
The study aims to preserve biological important microbial isolates such as plant growth promoters and biological control agents against rice pathogens developed through PhilRice- funded projects. Moreover, virulent isolates of major rice pathogens are also being preserved and maintained at the laboratory.
Highlights:
• One year in different storage conditions , two strains of a biocontrol agent, Trichoderma sp, and one isolate of a plant growth promoting bacteria, Streptomyces mutabilis, were evaluated in terms of culture viability, virulence and stability of their biologically important physiological traits.
• At one year in storage in different culture preservation conditions, fungal spores of Trichoderma sp. remained viable and maintained its inhibitory activity against sheath blight pathogen, Rhizoctonia solani. Viability and virulence of fungal cultures did not differ among different storage conditions (Figure 18). However, periodically subcultured Trichoderma sp.
has smaller mycelial colony size than those cultures stored in filter paper, mineral oil and 10% glycerol (Figure 19).
• At one year in storage in carbonized rice hull (CRH) and a soil-based carrier, cells of S. mutabilis remained viable and
colony count is comparable with the periodic subcultured samples. (Figure 20). However, colony count is lower when S.
mutabilis (either in CRH or soil-based carrier) is stored inside a refrigerator than left at room temperature. IAA production and ACC- deaminase activity of S. mutabilis were maintained regardless of the storage condition. (Figure 21 and Figure 22).
• For our collection of rice pathogens, differential isolates of rice blast (Pyricularia oryzae) are being maintained in filter paper and stored at -20ºC. Differential isolates of bacterial leaf blight pathogen, Xanthomonas oryzae, are stored in Microbank™ and in Wakimoto culture medium covered with 10% skim milk.
Sclerotial bodies of sheath blight pathogen, Rhizoctonia solani, are stored in microtubes and placed inside a refrigerator.
Cultures of bakanae pathogen, Fusarium moniliforme are maintained in potato dextrose agar slants and stored inside the refrigerator.
Figure 19. Dual culture assay between sheath blight pathogen, Rhizoctonia solani (right side of the plate) and Trichoderma sp. (left side of the plate) that has been subjected to different storage conditions. The microtubes containing fungal spores in filter paper, in 10% glycerol and in mineral oil were all stored in -80°C freezer for one year. The control has been regularly sub-cultured in PDA medium every 1 to 2 months.
Figure 20. Colony size of Trichoderma sp. after subjected to different storage conditions. Measurement was taken at 4 four days after placing spores of
Trchoderma sp in fresh PDA medium. The microtubes containing fungal spores in filter paper, in 10% glycerol and in mineral oil were all stored in -80°C freezer for one year. The control has been regularly sub-cultured in
PDA medium every 1-2 months.
Figure 21. Effect of different storage conditions on the population of Streptomyces mutabilis.
Figure 22. Effect of different storage conditions on the indole-3-acetic acid (IAA) production activity of Streptomyces mutabilis . To measure IAA production, test cultures were grown in arginine- glycerol- salt (AGS) broth supplemented wih tryptophan. After 7 days of incubation, the cultures were centrifuged and the IAA in supernatant was added with Fe-H2SO4 reagent.
Pink to red color indicated positive reaction. Test tube in extreme left , in each picture panel, is AGS broth only (negative control).
Figure 23. Effect of different storage conditions on the 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Streptomyces mutabilis . To test ACC-deaminase activity, the isolates were
grown using the nitrogen-free Dworkin and Foster’s salts minimal agar medium ( Dworkinand Foster, 1958). The plates were incubated at 28+/-2°C
in the dark for 7 days. Growth and sporulation of the isolates are indicators of ACC utilization and production of ACC deaminase
CRH in room temp. CRH in refrigerator Soil-based carrier in
room temp. Soil-based carrier in refrigerator
Abbreviations and acronymns
ABA – Abscicic acid Ac – anther culture AC – amylose content
AESA – Agro-ecosystems Analysis AEW – agricultural extension workers AG – anaerobic germination AIS – Agricultural Information System ANOVA – analysis of variance AON – advance observation nursery AT – agricultural technologist AYT – advanced yield trial BCA – biological control agent BLB – bacterial leaf blight BLS – bacterial leaf streak BPH – brown planthopper Bo - boron
BR – brown rice
BSWM – Bureau of Soils and Water Management
Ca - Calcium
CARP – Comprehensive Agrarian Reform Program
cav – cavan, usually 50 kg CBFM – community-based forestry management
CLSU – Central Luzon State University cm – centimeter
CMS – cystoplasmic male sterile CP – protein content
CRH – carbonized rice hull CTRHC – continuous-type rice hull carbonizer
CT – conventional tillage Cu – copper
DA – Department of Agriculture DA-RFU – Department of Agriculture- Regional Field Units
DAE – days after emergence DAS – days after seeding DAT – days after transplanting DBMS – database management system DDTK – disease diagnostic tool kit DENR – Department of Environment and Natural Resources
DH L– double haploid lines DRR – drought recovery rate DS – dry season
DSA - diversity and stress adaptation DSR – direct seeded rice
DUST – distinctness, uniformity and stability trial
DWSR – direct wet-seeded rice EGS – early generation screening EH – early heading
EMBI – effective microorganism-based inoculant
EPI – early panicle initiation ET – early tillering
FAO – Food and Agriculture Organization Fe – Iron
FFA – free fatty acid
FFP – farmer’s fertilizer practice FFS – farmers’ field school FGD – focus group discussion FI – farmer innovator
FSSP – Food Staples Self-sufficiency Plan g – gram
GAS – golden apple snail GC – gel consistency
GIS – geographic information system GHG – greenhouse gas
GLH – green leafhopper GPS – global positioning system GQ – grain quality
GUI – graphical user interface GWS – genomwide selection GYT – general yield trial h – hour
ha – hectare
HIP - high inorganic phosphate HPL – hybrid parental line I - intermediate
ICIS – International Crop Information System
ICT – information and communication technology
IMO – indigenous microorganism IF – inorganic fertilizer
INGER - International Network for Genetic Evaluation of Rice
IP – insect pest
IPDTK – insect pest diagnostic tool kit IPM – Integrated Pest Management IRRI – International Rice Research Institute IVC – in vitro culture
IVM – in vitro mutagenesis
IWM – integrated weed management JICA – Japan International Cooperation Agency
K – potassium kg – kilogram
KP – knowledge product
KSL – knowledge sharing and learning LCC – leaf color chart
LDIS – low-cost drip irrigation system LeD – leaf drying
LeR – leaf rolling lpa – low phytic acid LGU – local government unit
LSTD – location specific technology development
m – meter
MAS – marker-assisted selection MAT – Multi-Adaption Trial MC – moisture content
MDDST – modified dry direct seeding technique
MET – multi-environment trial MFE – male fertile environment MLM – mixed-effects linear model Mg – magnesium
Mn – Manganese
MDDST – Modified Dry Direct Seeding Technique
MOET – minus one element technique MR – moderately resistant
MRT – Mobile Rice TeknoKlinik MSE – male-sterile environment MT – minimum tillage mtha-¹ - metric ton per hectare MYT – multi-location yield trials N – nitrogen
NAFC – National Agricultural and Fishery Council
NBS – narrow brown spot
NCT – National Cooperative Testing NFA – National Food Authority NGO – non-government organization NE – natural enemies
NIL – near isogenic line NM – Nutrient Manager
NOPT – Nutrient Omission Plot Technique NR – new reagent
NSIC – National Seed Industry Council NSQCS – National Seed Quality Control Services
OF – organic fertilizer OFT – on-farm trial OM – organic matter ON – observational nursery
OPAg – Office of Provincial Agriculturist OpAPA – Open Academy for Philippine Agriculture
P – phosphorus PA – phytic acid
PCR – Polymerase chain reaction PDW – plant dry weight PF – participating farmer PFS – PalayCheck field school
PhilRice – Philippine Rice Research Institute PhilSCAT – Philippine-Sino Center for Agricultural Technology
PHilMech – Philippine Center for Postharvest Development and Mechanization
PCA – principal component analysis
PI – panicle initiation PN – pedigree nursery
PRKB – Pinoy Rice Knowledge Bank PTD – participatory technology development
PYT – preliminary yield trial QTL – quantitative trait loci R - resistant
RBB – rice black bug
RCBD – randomized complete block design RDI – regulated deficit irrigation
RF – rainfed RP – resource person RPM – revolution per minute
RQCS – Rice Quality Classification Software RS4D – Rice Science for Development RSO – rice sufficiency officer RFL – Rainfed lowland RTV – rice tungro virus
RTWG – Rice Technical Working Group S – sulfur
SACLOB – Sealed Storage Enclosure for Rice Seeds
SALT – Sloping Agricultural Land Technology SB – sheath blight
SFR – small farm reservoir SME – small-medium enterprise SMS – short message service SN – source nursery
SSNM – site-specific nutrient management SSR – simple sequence repeat
STK – soil test kit
STR – sequence tandem repeat SV – seedling vigor
t – ton
TCN – testcross nursery
TCP – technical cooperation project TGMS – thermo-sensitive genetic male sterile
TN – testcross nursery TOT – training of trainers TPR – transplanted rice TRV – traditional variety TSS – total soluble solid UEM – ultra-early maturing
UPLB – University of the Philippines Los Baños
VSU – Visayas State University WBPH – white-backed planthopper WEPP – water erosion prediction project WHC – water holding capacity WHO – World Health Organization WS – wet season
WT – weed tolerance YA – yield advantage Zn – zinc
ZT – zero tillage